Nucleophilic polysubstituted aryl acridinium ester conjugates and syntheses thereof

ABSTRACT

This invention is directed to novel nucleophilic polysubstituted aryl acridinium conjugates and the methods for preparation thereof. The novel nucleophilic polysubstituted aryl acridinium conjugates are useful in biological assays, including novel assays for the determination of Vitamin B12, folate, cortisol, estradiol, and thromboxane B2.

This application is a continuation of application Ser. No. 08/032,947filed on Mar. 17, 1993 (now U.S. Pat. No. 5,663,074) which is aContinuation in part of application Ser. No. 07/871,601 filed Apr. 17,1992 (now U.S. Pat. No. 5,241,070) which is a Continuation ofapplication Ser. No. 07/249,620 filed Sep. 26, 1988, now abandoned.

FIELD OF THE INVENTION

This invention relates to novel nucleophilic polysubstituted arylacridinium esters. This invention also relates to conjugates formed fromthe novel nucleophilic polysubstituted aryl acridinium esters. Thisinvention further relates to assays utilizing the novel nucleophilicpolysubstituted aryl acridinium esters and conjugates thereof.

BACKGROUND OF THE INVENTION

The use of acridinium esters as chemiluminescent labels in clinicalassays is known. For example, European Patent Application No. 82306557.8describes the use of an aryl acridinium ester linked to anN-succinimidyl moiety as a chemiluminescent label in immunoassays. U.S.Pat. No. 4,745,181 and copending U.S. patent application Ser. No.133,792, filed Dec. 14, 1987, now U.S. Pat. No. 4,918,192 describepolysubstituted aryl acridinium esters which are useful in immunoassaysand nucleic acid hybridization assays. U.S. patent application Ser. No.226,639, filed on Aug. 1, 1988, describes hydrophilic polysubstitutedaryl acridinium esters and conjugates thereof useful in clinical assays,particularly those assays involving liposomes.

Richardson et al (Clin. Chem. 31/10, 1664-1668, 1985) and Miller et al(Ann. Clin. Biochem 25, 27-34, 1988) describe the use of4-(2-aminoethyl)phenyl acridine-9-carboxylate in a chemiluminescentimmunoassay for plasma progesterone. However, the prior art acridiniumesters often cannot effectively form conjugates with certain analytes.These analytes may lack nucleophilic groups or may contain carboxylicgroups which are not readily amenable to modifications. In some cases,the nucleophilic group of the analyte cannot be acylated with anactive-group containing acridinium ester because of the resultingdeleterious effect on the immunoactivity of the analyte.

The acridine ester of Richardson et al and Miller et al cannot beconverted to a useful acridinium ester without the concurrent loss ofthe nucleophilicity of the ester. The acridine ester must be conjugatedfirst with the target analyte through a nucleophilic reaction, and thensubsequently converted to an acridinium ester moiety. The generalreaction conditions of the acridine ester-to-acridinium ester conversionare non-selective. As a result, susceptible groups on the targetanalytes are frequently affected, resulting in the loss of or reductionof the immunoactivity of the resulting conjugate.

Accordingly, it is the purpose of the present invention to provide novelnucleophilic polysubstituted aryl acridinium esters. It is also apurpose of the present invention to provide novel nucleophilicpolysubstituted aryl acridinium esters which contain an additionalionizable group.

It is a further purpose of this invention to provide conjugates formedfrom the novel nucleophilic polysubstituted aryl acridinium esters.

It is a still further purpose of this invention to provide assaysutilizing the novel nucleophilic polysubstituted aryl acridinium estersand conjugates thereof.

DESCRIPTION OF THE INVENTION

This invention relates to nucleophilic polysubstituted aryl acridiniumesters of the formula: ##STR1## wherein R₁ is alkyl, alkenyl, alkynyl,aryl, or aralkyl containing from 0 to 20 heteroatoms, preferablynitrogen, oxygen, phosphorous, or sulfur;

R₂, R₃, R₅, and R₇ are hydrogen, amino, alkoxyl (--OR), hydroxyl,--COOH, halide, nitro, --CN, --SO₃ H, ##STR2## --SCN, --R, --SR, or--SSR, wherein R is alkyl, alkenyl, alkynyl, aryl, or aralkyl,containing from 0-20 heteroatoms;

R₄ and R₈ are alkyl, alkenyl, alkynyl, aralkyl, or alkoxyl;

X is an anion, preferably CH₃ SO₄ ⁻, FSO₃ ⁻, halide, CF₃ SOR₃ ⁻, C₄ F₉SO₃ ⁻, or ##STR3## and

R₆ is: Q--R--Nu, ##STR4## or Q--Nu wherein Q is --O--, --S--, --NH--,##STR5## diazo, or ##STR6## R is as defined above; I is --SO₃ H, --OSO₃H, --PO(OH)₂, --OPO(OH)₂, or --COOH; and Nu is a nucleophilic group.

A nucleophilic group for the purpose of this invention is defined as achemical group which is electron rich, has an unshared pair of electronsacting as a reactive site, and seeks a positively charged orelectron-deficient site on another molecule. Examples of usefulnucleophilic groups include amino, hydroxyl, sulfhydryl, or an activemethylene group, adjacent to a strong electron-withdrawing group. Astrong electron-withdrawing group is defined as a chemical group orsubstituent which strongly attracts electrons and, therefore,intensifies the positive charge of the carbon atom (or carbonium ion) ornullifies the negative charge of the carbon atom (or carbanion), towhich the group is attached. Examples of strong electron-withdrawinggroups include --NO₂, --CN, --SO₃ H, --N(R)₃ ⁺, --S(R)₂ ⁺, and --NH₃ ⁺,wherein R is as defined above.

Organic metallic moieties are also useful nucleophilic groups for thepurposes of this invention. An organic metallic moiety is defined as anorganic moiety comprising carbon-metal bonds. Examples of organicmetallic moieties include Grignard reagents, lithium compounds, andphenylsodium.

Preferably R₁ is alkyl, alkenyl, alkynyl, aryl, or aralkyl of from 1 to24 qarbon atoms;

R₂, R₃, R₅, and R₇ are hydrogen, amino, --COOH, --CN, hydroxyl, alkoxylof from 1 to 4 carbon atoms, nitro, halide, --SO₃ H, or --SCN;

R₄ and R₈ are alkyl, alkenyl, alkynyl, or alkoxyl, of from 1 to 8 carbonatoms; and X is halide.

More preferably, R₁ is alkyl of from 1 to 10 carbon atoms; R₂, R₃, R₅,and R₇ are hydrogen, nitro, --CN, halide, alkoxyl of from 1 to 4 carbonatoms, amino, or --SO₃ H; and R₄ and R₈ are alkyl of from 1 to 4 carbonatoms.

Most preferably, R₁, R₄, and R₈ are methyl; R₂, R₃, R₅, and R₇ arehydrogen; X is bromide; and R₆ is --CONH--CH₂ CH₂ --NH₂ or ##STR7##

The R₅ and R₆ position can be interchanged in the acridinium esters ofthis invention. Accordingly, the preferred acridinium esters of thisinvention include acridinium esters of the following formula: ##STR8##wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and X are as defined above.

This invention also relates to conjugates comprising the above-describedacridinium esters of this invention covalently bound to any compoundwhich can covalently bind to the Nu group of the acridinium ester. Toform a useful conjugate, the compound must contain at least onefunctional group capable of binding to the Nu group of the acridiniumester. Preferably, the conjugates are formed from compounds havingbiological activity, such as proteins, nucleic acids, antigens, haptens,etc.

If Nu is NH₂, for example, examples of suitable compounds for formingconjugates of this invention include those compounds which containfunctional groups capable of binding with --NH₂, such as:

(1) carboxylate groups, as in, e.g., folic acid, carboxylated VitaminB₁₂, Vitamin B₁₂ -hemisuccinate at the ribose moiety, N-hemisuccinatesof T₄ -methyl ester and T₃ methyl ester, thromboxane B₂,carboxypropyl-theophylline, penicillins, cortisol-3-carboxylmethyloxime,estradiol-6-carboxymethyloxime, morphine-6-hemisuccinate, and the like;(2) ketone groups, as in, e.g., 3-ketodigoxigenine; (3) aldehyde groups,as in, e.g., digoxin-dialdehyde and bromouridine dialdehyde; (4)halides, as in, e.g., dinitroflurobenzene and chlorotriazine derivativesof haptens and proteins; (5) active esters, as in, e.g.,N-hydroxysuccinimide and imidate derivatives of haptens and proteins;(6) isocyanate and thioisocyanate, as in, e.g., hapten and proteinderivatives.

If Nu is --SH, examples of suitable compounds will contain functionalgroups capable of binding with --SH, such as maleimido, dithiopyridino,or olefin as found in, e.g., hapten and protein derivatives.

If Nu is --OH, examples of suitable compounds for forming the conjugatesof this invention include those compounds which contain functionalgroups capable of binding with --OH, such as oxirane, as found in, e.g.,hapten and protein derivatives.

If Nu is a Grignard moiety or other organo-metallic moiety, examples ofsuitable compounds for forming the conjugates of this invention includethose compounds which contain functional groups capable of binding tothe moiety, such as ketone and aldehyde, as found in, e.g., aprotichaptens.

It will be appreciated that numerous other suitable Nu groups can beutilized in the acridinium esters of this invention. It is left for theartisan to choose, which combination of acridinium ester and conjugatingcompound best serves the needs of the desired application.

The term "activation" (or activate) for the purposes of thespecification and the claims means a modification of an existingfunctional group of a particular compound, which modification generates(or introduces) a new reactive functional group from the prior existingfunctional group, which new reactive functional group is capable ofbinding to a target functional group of a second compound. For example,the carboxylic group (--COOH) in thromboxane B₂ (see structure below)can be "activated" to produce a mixed anhydride group ##STR9## usingknown procedures in the art. The mixed anhydride can then react with theamino group (--NH₂), for example, of 2', 6'-dimethyl-4'-[N-(2aminoethyl)carbomoyl]phenyl 10-methylacridinium-9-carboxylate bromide(DMAE-ED), to form an amide linkage ##STR10## resulting in the formationof a Thromboxane B₂ -DMAE-ED conjugate (see Example 11 below). As anadditional example, the free amino group (--NH₂) group on the surface ofalkyl siloxane-coated paramagnetic particles (PMP) (Advanced MagneticsInc., Cambridge, Mass.) can be "activated" by derivatization withhomobifunctional glutaraldehyde ##STR11##

One reactive aldehyde group of the glutaraldehyde covalently binds tothe PMP by formation of a Schiff base with the free amino group. Thesecond reactive aldehyde group can then bind with a protein.

The preparation of the conjugates of this invention will vary dependingon the acridinium ester and conjugating compound chosen. For example,the following discussion will highlight certain exemplary approaches toforming conjugates from certain compounds and certain preferred Nugroups on the acridinium esters of this invention: (1) when Nu is --NH₂and the conjugating compound contains a carboxylic group, the carboxylicacid group is first activated to form an active ester, such asN-hydroxysuccinimide ester, mixed anhydride, or acyl halide. Theactivated compound is then reacted with the acridinium ester to form theconjugate; (2) when Nu is --NH₂ and the conjugating compound contains aketone or aldehyde group, the acridinium ester can be directly reactedwith the compound to form a Schiff base. The conjugate can then bereacted with a hydride reducing agent, such as sodium cyanoborohydride,to stabilize the linkage; (3) when Nu is --NH₂, the acridinium ester canbe reacted directly with a conjugating compound containing a reactivegroup like halide, isocyanate, or thioisocyanate; (4) when Nu is --SH,the conjugating compound should contain a thiol (sulfhydryl)-reactivegroup, such as maleimido, dithiopyridino, or olefin, to effectivelyreact with the acridinium ester to form a conjugate; (5) when Nu is--OH, it is preferred that the desired conjugating compound contain anoxirane group to effectively react with the acridinium ester to form aconjugate; (6) when Nu is a Grignard or other organo-metallic moiety,the acridinium ester containing such a moiety should be prepared freshfor each use and then reacted with a conjugating compound containing aketone or aldehyde functional group to form the conjugate.

It will be appreciated that the discussion above is not exhaustive andthat numerous other conjugates can be formed from the novel acridiniumesters of this invention using known procedures in the art.

The conjugates of this invention are useful as luminescent tracers. Theconjugates are particularly useful in luminescent assays using specificbinding phenomena such as antibody/antigen immunological reactions,nucleic acid hybridization reactions, or ligand/binding proteininteractions.

In one embodiment of the present invention, conjugates are preparedusing the acridinium esters of this invention and folate or folatederivatives. Preferably, the acridinium ester used contains bothnucleophilic and hydrophilic groups. Preparation of thisfolate-acridinium ester conjugate involves incubating the acridiniumester with a protected folate intermediate of the following formula(which can be activated at one or both of its carboxylic groups):##STR12## wherein R' is an optional branched or straight-chain,saturated or unsaturated, alkyl of from 1 to 24 carbon atoms, containing0-20 heteroatoms, preferably nitrogen, oxygen, phosphorous, or sulfur.R" is Z₂, hydrogen, or a branched or straight-chain, saturated orunsaturated, alkyl of from 1 to 24 carbon atoms, containing 0-20heteroatoms, preferably nitrogen, oxygen, phosphorous, or sulfur. Thedotted lines are optional double bonds.

Z₁ and Z₂ are protecting groups. Z₂ is optional. The protecting groupscan be any group which can protect the primary and secondary amines fromreacting with the activated carboxylic group of the folate either intra-or inter-molecularly. The protecting groups must be removable underconditions which do not deleteriously affect the acridinium ester,preferably in an acidic environment. Useful protecting groups includetrifluoroacetyl or t-butyloxycarbonyl groups.

After conjugation of the protected folate intermediate with theacridinium ester, the folate moiety is deblocked by removal of theprotecting groups. This deblocking is preferably conducted in an acidicenvironment using an acidic media such as HBr/acetic acid, which iscapable of removing the protecting groups without destroying theintegrity of the conjugate. The conjugate so formed can then be used asa tracer in an assay for measuring folates.

In another embodiment of this invention, conjugates are formed using theacridinium esters of this invention and Vitamin B₁₂ (cyanocobalamin).Vitamin B₁₂ has the following structure: ##STR13##

Treating Vitamin B₁₂ with dilute acid will deaminate 1, 2 and/or 3 ofthe primary propanamide side chains of the Corrin ring to generate acarboxylic function. This carboxylate function is then used to conjugatethe Vitamin B₁₂ to the nucleophilic acridinium esters of this invention.

The ratio of monocarboxylic Vitamin B₁₂ (one carboxylate function),dicarboxylic Vitamin B₁₂ (two carboxylate functions), and tricarboxylicVitamin B₁₂ (three carboxylate functions) will depend on the acidconcentration and the reaction time. Monocarboxylic Vitamin B₁₂ is thedesired product for the purpose of preparing the conjugates of thisinvention. Typically, by optimizing known procedures, such as theprocedure described in Allen et al, J. Biol. Chem. 247, 7695 (1972),mixtures of mono-, di-, and tricarboxylic Vitamin B₁₂ can be generatedwhich contain up to 40% monocarboxylic Vitamin B₁₂ (see FIGS. 3 and 4).Prior art procedures utilize a strong anion exchanger to separate themonocarboxylic Vitamin B₁₂ from the di- and tricarboxylic Vitamin B₁₂.[Allen et al, J. Biol. Chem. 247, 7695, 1972.)

There are potentially three forms of monocarboxylic Vitamin B₁₂,depending on which of the 3 primary propanamide side chains have beendeaminated. It is desirable to separate these three monocarboxylicVitamin B₁₂ forms.

It has been unexpectedly discovered that by separating the mixture ofcarboxylated Vitamin B₁₂ on Reverse Phase High Perfomance LiquidChromatography (HPLC), the monocarboxylic Vitamin B₁₂ forms can beseparated and isolated from the di- and tri-carboxylic forms and fromeach other. Accordingly, this allows the artisan to obtain individualmonocarboxylic Vitamin B₁₂ forms with high purity without thepreliminary ion-exchange fractionation step of the prior art.

It has also been unexpectedly discovered that one monocarboxylic VitaminB₁₂ form is more effective than the other two forms in the conjugates ofthis invention for use in Vitamin B₁₂ assays.

In a further embodiment of this invention, conjugates are formed usingthe acridinium esters of this invention and estradiol. These conjugatescan be used, for example, as tracers in assays for 17-beta-estradiol.

17-beta-estradiol has the following structure: ##STR14## wherein A isCH₂. Useful derivatives of 17-beta-estradiol include6-keto-17-beta-estradiol (where A is ##STR15## and, preferably,6-carboxymethyloxime-17-beta-estradiol (where A is ##STR16##

Conjugation with an appropriate acridinium ester of this invention canoccur at any of the available functional groups on th e17-beta-estradiol or derivative therefore, i.e., the phenolic 3-OHgroup, the secondary 17-OH group, or the keto or the carboxymethyloximegroup created at the C-6 position. The choice of functional group forconjugation will depend generally on such factors as compatibility withthe desired immunoassay system, the stability of conjugate prepared, andthe ease of preparation. Preferably the conjugate is prepared byactivating the carboxylic group of6-carboxymethyloxime-17-beta-estradiol and then reacting the activatedestradiol derivative with an appropriate acridinium ester-of theinvention. The resulting conjugate can then be used as a tracer in anassay for determining 17-beta-estradiol.

The acridinium esters of this invention can also be used to form usefulconjugates with cortisol. Cortisol has the following structure:##STR17## wherein B is ##STR18## A derivative of cortisol is3-carboxymethyloxime cortisol (where B is ##STR19##

Conjugation with an appropriate acridinium ester of this invention canoccur at any of the available functional groups on the cortisol orderivative thereof, i.e., the 21-OH group, the 17-OH group, the 11-OHgroup, the 20-keto group, the 3-keto group, and the 3-carboxymethyloximegroup. Preferably, the conjugate is prepared by activating thecarboxylic group of the 3-carboxymethyloxime cortisol and then reactingthe activated cortisol derivative with an appropriate acridinium esterof this invention. The resulting tracer can then be used as a tracer inan assay for cortisol.

Useful conjugates can be formed between the acridinium esters of thisinvention and thromboxane B₂ and other prostaglandin analogs.Thromboxane B₂ has the following structure: ##STR20##

Conjugation with an appropriate acridinium ester of this invention canoccur at any of the three hydroxyl groups or the olefin functionalgroups. The choice of functional group will depend generally on suchfactors as compatibility with a specific binding protein, and low crossreactivity with non-target prostaglandin analogs. Preferably, theconjugate is prepared by activating the terminal carboxylic group of thethromboxane B₂ and then reacting the resulting thromboxane B₂ derivativewith an appropriate acridinium ester of this invention. The resultingconjugate can then be used as a tracer in assays for thromboxane B₂.

This invention also relates to assays utilizing the conjugates of thisinvention as chemiluminescent tracer compounds. The assays can behomogeneous or heterogeneous. The assays can be competitive inhibitionassays where, for example, the analyte to be determined is a univalenthapten molecule. The assays can also be non-competititve, such assandwich assays where, for example, the acridinium esters of thisinvention are conjugated to an antibody or a receptor molecule. Thecomponents or reagents of the assays utilizing the conjugates of thisinvention can be mixed together in any desired manner or sequenceprovided that the resultant acridinium ester label can be measured in asubsequent detection system. Accordingly, the assays utilizing theconjugates of this invention can be conducted in a forward mode, reversemode, or a simultaneous mode (see, e.g., U.S. Pat. Nos. 4,098,876 and4,244,940).

Assays for the detection and measurement of Vitamin B₁₂ and folate areillustrative of the assays which can be conducted using the conjugatesof this invention. Such assays can use the Vitamin B₁₂ -aqridinium esteror the folate-acridinium ester conjugates of this invention. A generaldiscussion of radioisotope dilution assays for Vitamin B₁₂ and forfolate is found in U.S. Pat. No. 4,451,571, herein incorporated byreference.

Assays for the detection or measurement of Vitamin B₁₂ or folate in asample generally require a sample preparation step wherein the VitaminB₁₂ or folate in the sample is released (liberated) from endogenousbinding proteins. Methods to release the Vitamin B₁₂ or folate from therespective binding proteins include heating or boiling the sample, orusing a chemical releasing agent. Typical releasing agents comprise astrong base, such as NaOH. A sulfhydryl compound, such as dithiothreitol(DTT) or beta-mercaptoethanol, is also typically added during the samplepreparation step. The sulfhydryl compound can be added before, after, oralong with, the addition of the releasing agent.

In one assay for Vitamin B₁₂, following the sample preparation step, theVitamin B₁₂ tracer compound is combined with the sample and purified hogintrinsic factor (HIF) immobilized on a solid phase. The sample andtracer compound compete for binding sites on the HIF. The amount oftracer compound bound to HIF is inversely proportional to the amount ofVitamin B₁₂ in the sample.

In one assay for folate, following the sample preparation step, thefolate tracer compound is combined with the sample and bovinelactoglobulin or folate binding protein (FBP), immobilized on a solidphase. The sample and tracer compound compete for binding sites on theFBP. The amount of tracer compound bound to FBP is inverselyproportional to the amount of folate in the sample.

It has been discovered that the sulfhydryl compounds used in the samplepreparation step remain in the solid phase at the time of counting(i.e., measuring the amount of label associated with the solid phase).The presence of varying concentrations of the sulfhydryl compound(particularly DTT) in the solid phase can quench the photon output ofthe chemiluminescent reaction of the acridinium ester label and resultin poor assay precision and reduced signal. It was unexpectedlydiscovered that by incubating the solid phase in a solution comprising athiol-reactive compound, such as ethyl maleimide, prior to counting, thequenching effect of the sulfhydryl compound is reduced or eliminated.Preferably, the concentration of the thiol-reactive compound in thesolution is about 0.01 mM to about 50 mM, more preferably about 0.5 mMto about 10 mM, and most preferably, about 1 mM.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-6 are profiles of the separation of mixtures containingcarboxylated Vitamin B₁₂ using HPLC and are discussed in Example 5.

FIGS. 7-13 represent the assay performance in competitive immunoassaysof different conjugates (traces) formed between analytes andnucleophilic polysubstituted aryl acridinium esters. These Figures arediscussed in Examples 6 and 12-16.

The following Examples illustrate the present invention.

EXAMPLE 1 Preparation of2',6'-Dimethyl-4'-[N-(2-aminoethyl)carbomoyl]phenyl10-methylacridinium-9-carboxylate bromide (DMAE-ED)

A solution of 2',6'-dimethyl-4'-carboxylphenyl10-methylacridinium-9-carboxylate bromide (DMAE, 200 mg, 0.43 mmole)(see copending U.S. application Ser. No. 226,639, filed on Aug. 1, 1988)in 30 ml of dimethylformamide (DMF) was cooled in ice bath, treated withtriethylamine (0.25 ml, 1.72 mmole), ethylchloroformate (0.08 ml, 0.85mmole) and 30 ml of chloroform to form a reaction mixture. After 20 min.of stirring, the reaction mixture was transferred to a dried droppingfunnel and added dropwise over a 15 minute period to a solution ofethylenediamine in 10 ml of DMF/CHCl₃ (1:1) to form a second reactionmixture.

The second reaction mixture was then stirred at room temperatureovernight and then evaporated to dryness under vacuo. The residueproduced from the evaporation was taken up in 3-4 ml ofchloroform/methanol/water (65:25:4), purified on two 20×20 cmpreparative thin layer chromatography TLC plates (Silica gel 60, F254,Merck & Co., Inc., Rahway, N.J.) and developed with the same solventsystem. The yellow major band which developed (Rf=0.47) (which couldalso be detected under long and short UW light) was stripped and elutedwith the same solvent system. The eluent was then evaporated. Theresidue from this evaporation was triturated with 30 ml of 10%methanol/chloroform and filtered through Whatman #1 filter paper undergravity. The filtrate so produced was evaporated to produce DMAE-ED (110mg, 50%). Fast Atom Bombardment (FAB) Mass Spectral Analysis (performedby Oneida Research Services, Whitesboro, N.Y.) in the positive ion modegave a M+ peak of 428. Isotopic bromide peaks 79 and 81 of about equalintensity were detected in the negative ion mode.

EXAMPLE 2 Preparation ofN-tert-Butyloxycarbonyl-S-(3-sulfopropyl)cysteine (BOC-SulfoCys)

2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (BOC-ON) (121 mg,0.49 mmole) (Aldrich Chem. Co., Milwaukee, Wis.) was added to a solutionof S-(3-sulfopropyl)cysteine (SulfoCys, 100 mg, 0.41 mmole), (preparedby the method of U. T. Ruegg and J. Rudinger, J. Peptide Protein Res. 6,447, 1974), and triethylamine (0.18 ml, 1.23 mmole) in 3 ml of 50%aqueous dioxane to form a reaction mixture. The reaction mixture washeated at 50°-55° C. for 1 hour to obtain a yellow solution. Thereaction mixture was then cooled, diluted with 5 ml of water, and washedwith ethylacetate (3×10 ml). The resultant aqueous layer was evaporatedto dryness under vacuo by coevaporating with methanol twice. TLC (Silicagel 60, Merck & Co., Inc,) analysis of the residue produced by theevaporation, using a solvent system of chloroform/methanol/water(55:40:5), showed complete conversion of SulfoCys to BOC-SulfoCys.

EXAMPLE 3 Preparation of2',6'-Dimethyl-4'-{N-[N-(2-tert-butyloxycarbonylamino-3-S-(3'-sulfopropyl)-thiopropionyl)-2-aminoethyl]carbamoyl}phenyl10-methylacridinium-9-carboxylate bromide (BOC-SulfoCys-ED-DMAE)

A solution of BOC-SulfoCys (3.42 mmole) (Example 2), and DMAE-ED (400mg, 0.79 mmole) (Example 1) in 110 ml of DMF/CHCl₃ (1:1) was treatedwith dicyclohexylcarbodiimide (325 mg, 1.57 mmole), stirred at roomtemperature for 3 hours and evaporated to dryness. The residue from theevaporation was taken up in about 15 ml of chloroform/methanol/water(65:25:4) and purified on 8 preparative TLC plates (Silica gel 60, Merck& Co., Inc.) developed with the same solvent system.

The major yellow band which developed at about Rf of 0.55 was strippedand eluted with the same solvent system. The eluent was then evaporatedto dryness under vacuo to produce BOC-SulfoCys-ED-DMAE (630 mg, 90%)

EXAMPLE 4 Preparation of2',6'-Dimethyl-4'-{N-[N-(2-amino-3-S-(3'-sulfopropyl)-thiopropionyl)-2-aminoethyl]carbamoyl}phenyl10-methylacridinium-9-carboxylate bromide (SulfoCys-ED-DMAE)

A solution of BOC-SulfoCys-ED-DMAE (630 mg, 0.715 mmole) (Example 3) in4 ml of 36% HBr/Acetic acid was kept at room temperature for 5 hours toform a reaction mixture. The reaction mixture was added dropwise toabout 30 ml of anhydrous ethylether, forming a gummy precipitate andsupernatant. The supernatant was removed from the precipitate. Theprecipitate was then dissolved in about 10 ml of methanol and theresultant solution was then added dropwise to about 30 ml of freshanhydrous ethylether, forming a yellow precipitate and supernatant.

The yellow precipitate and supernatant were then filtered through amedium porosity frit and the resultant yellow solid residue was thenwashed with anhydrous ethylether, and then air dried to produceSulfoCys-ED-DMAE (438 mg, 93.7%).

FAB Mass Spectral analysis (performed by both Oneida Research Services,Whitesboro, N.Y. and Institute of Chemical Analysis, NortheasternUniversity, Boston, Mass.) in the positive ion mode gave a M+ peak of653.

EXAMPLE 5 Preparation of Monocarboxylic Vitamin B₁₂

A. Preparation of Deaminated Vitamin B₁₂

Vitamin B₁₂ (1.0 g, 0.738 mmole) (Sigma Chemicals, St. Louis, Mo.) wasadded to 80 ml of 0.5N HCl to form a reaction mixture and stirred atroom temperature for 65 hours. The reaction mixture was then loaded ontoa 4×5 cm column of Bio-Rad AG1-X8 (acetate form) (Bio-Rad Laboratories,Richmond, Calif.), 100-200 mesh, packed and eluted as described inAllen, R. H. and Majerus, P. W., J. Biological Chem., 247, 7695-7701(1972). The initial 300 ml of red eluent was collected and evaporated todryness under vacuum to produce about 1 gram of a mixture of mono-, di-,and tricarboxylated Vitamin B₁₂.

B. Preparation of Mixture of Mono-carboxylated Vitamin B₁₂

The mixture of carboxylated Vitamin B₁₂ prepared in A was fractionatedby QAE-Sephadex A-25 chromatography to obtain a mixture ofmonocarboxylated Vitamin B₁₂ as described by Allen et al. (J. Biol.Chem. 247, 7695, 1972).

C. Preparative HPLC for the Separation of the Carboxylated Vitamin B₁₂

50 mg of the mixture of the mono-, di-, and tri-carboxylated Vitamin B₁₂from A in 2 ml of water was injected into a Waters Delta-Prep 3000 HPLCsystem (Waters Associates, Milford, Mass.) with an ISCO-Foxy fractioncollector, an ISCO-2150 peak separator (ISCO, Lincoln, Nebr.) and a YMCAP 363-15, 30 mm×25 cm stainless steel column packed with C18-bondedsilica of 15 um particles, spherical shape, and 300A pore size (YMC,Inc., Morris Plains, N.J.).

The carboxylated Vitamin B12 was eluted from the column for each runusing acetonitrile as Solvent B and 0.05M triethylammonium acetate, pH4.5, as Solvent A, in the following manner:

1. Run 1--step-gradient elution:

20 min. on 8% Solvent B/92% Solvent A then

10 min. on 10% Solvent B/90% Solvent A then

50 min. on 15% Solvent B/85% Solvent A

2. Run 2--isocratic elation:

Using 15% Solvent B/85% Solvent A

3. Run 3--step-gradient elution:

10 min. on 10% Solvent B/90% Solvent A

30 min. on 15% Solvent B/85% Solvent A

4. Run 4--isocratic elution:

10% Solvent B/90% Solvent A

The flow rate of the column was 20 ml/min. for each run and the elutedmaterials were detected at a wavelength of 280 nm.

FIG. 1 is a profile of the separation of the mixture of carboxylatedVitamin B₁₂ obtained in Run 1. Five peaks were collected although Peak 5was not recorded because the preprogrammed recording time ended prior toelution of Peak 5.

Peak 5 was determined based on the characteristic red color of thefraction collected. Peak 5 was then added to the graph as a dotted linepeak indicating its location had it been recorded.

FIGS. 2-4 are profiles of the separation of the mixture of carboxylatedVitamin B₁₂ obtained in Runs 4, 2, and 3, respectively.

These results demonstrate that with a properly chosen solvent system andpreparative HPLC column, the separation profile of the mixture of thecarboxylated Vitamin B₁₂ can be adjusted. For example, as seen in FIG. 2and FIG. 5A which show the profile of the analytical HPLC of the samemixture of carboxylated Vitamin B₁₂ (See D below), Peak 3 of FIG. 5A(retention time of 13.84 min.) was split into peaks 3 and 4 (FIG. 2)using the preparative column.

D. Analytical HPLC Profile of the Carboxylated Vitamin B₁₂ Derivatives

5 to 20 ug of the mixtures of carboxylated Vitamin B₁₂ prepared in A, B,and Run 2 of C, in 20 ul of water were each injected into a Beckman 344HPLC system (Beckman Instruments, San Ramon, Calif.). The HPLC systemcontained a 3.9 mm×30 cm stainless steel column packed with uBondapakC18 10 um particles with irregular shape and 120A pore size (WatersAssociates, Milford, Mass.). The mixtures were each eluted isocraticlyfrom the column using 10% acetonitrile and 90% 0.05M triethylammoniumacetate, pH4.5. The flow rate of the elution was 1.5 ml/min. and theeluted materials were detected at a wavelength of 280 nm.

FIG. 5A is a profile of the separation of the carboxylated Vitamin B₁₂from A. Peak 1 represents the unreacted Vitamin B₁₂, Peak 2 (retentiontime of 11.97 min.) and peak 3 (retention time of 13.84 min.) representthe 3 incompletely separated monocarboxylated Vitamin B₁₂ forms presentin the mixture. Peak 5 in FIG. 5A probably represents the dicarboxylatedVitamin B₁₂ form.

FIGS. 5B-5F are the analytical HPLC profiles of the separated peaks 1-5(FIG. 2) obtained in Run 2 of C. FIG. 5D and FIG. 5E show the closeretention times (13.54 min. and 14.33 min., respectively) of the twodifferent monocarboxylated Vitamin B₁₂ forms which comprised peak 3 ofFIG. 5A and peaks 3 and 4 of FIG. 2.

FIG. 6 is the analytical HPLC profile of the mixture of the 3monocarboxylated Vitamin B₁₂ forms from B. Peaks 12.22 min. and 13.9min. of FIG. 6 have nearly the same retention times as Peaks 2 and 3 ofFIG. 5A.

EXAMPLE 6 Preparation of Conjugates from Monocarboxylic Vitamin B₁₂ andDMAE-ED (B₁₂ -ED-DMAE)

Using the 3 monocarboxylic Vitamin B₁₂ forms prepared and isolated inExample 5 (designated hereinafter as monocarboxylic Vitamin B₁₂ forms I,II, and III, which have retention times of 11.58, 13.54, and 14.33 min.,respectively) and DMAE-ED prepared in Example 1, three separate butsimilar conjugating reactions were carried out to prepare the tracers asfollowing:

A solution of monocarboxylic Vitamin B₁₂ form I (10 mg., 7.4 umole) in1.8 ml of DMF was cooled in ice bath, treated with triethylamine (10.5ul, 74 umole, in 100 ul DMF) and ethyl chloroformate (2.8 ul, 30 umole,in 100 ul DMF) to form a reaction mixture. After stirring for 30minutes, the reaction mixture was evaporated to dryness to remove theexcess ethyl chloroformate to produce a residue. DMAE-ED (3.4 mg, 6.7umole) and triethylamine (5.2 ul, 37 umole) in 2 ml of DMF, were addedto the residue to form a second reaction mixture. The second reactionmixture was stirred at room temperature overnight and then evaporated todryness under vacuo. The crude products so obtained were purified on oneanalytical silica gel 20×20 cm TLC plate (Silica gel 60, Merck & Co,Inc.), developed with chloroform/methanol/water (55:40:5).

Two red bands (hereinafter referred to as the upper and the lower bands)which developed between Rf of 0.47-0.57 were each separately strippedand eluted with the same solvent system. Each eluent was evaporated todryness to produce a B₁₂ -ED-DMAE tracer.

The same procedure described above was repeated for monocarboxylicVitamin B₁₂ forms II and III. As a result, from the 3 monocarboxylicVitamin B₁₂ forms, a total of six B₁₂ -ED-DMAE conjugates (designated 1through 6) were isolated. Conjugates 1 and 2 were prepared from form I,3 and 4 were prepared from form II, and 5 and 6 were prepared from formIII. The conjugates were each diluted in phosphate buffered saline (PBS)with 0.1% bovine serum albumin (BSA) and were simultaneously screenedfor tracer activity (FIG. 7) using the following procedure:

A series of Vitamin B₁₂ standards in 1% human serum albumin (HSA) (inl2OmM PBS containing 0.2% sodium azide and 0.4 g/l merthiolate) weretreated by adding 1/20 volume of 1.35M DTT, to produce treatedstandards. 100 ul of each treated standard was added to a 12×75 mmplastic tube. To each tube was then added 100 ul of 0.5N NaOH and 0.5 mlof an IF-PMP (100 ug) (prepared as described in Example 12A below exceptthat the suspension contained 3 ug IF/g PMP, the heat stress step wasomitted, and the IF-PMP was resuspended in 0.16M boric acid, 10 mMphosphate, 0.127M NaCl, and 0.1% sodium azide, pH 7.0). 10 ul of PBS/BSAcontaining 12-29×10⁶ relative light units (RLU) (1 RLU=1 photon count)of the B₁₂ -ED-DMAE conjugate to be tested, was added to each tube andthe tubes were then incubated at room temperature for one hour. Thesolid phase in each tube was magnetically separated from the supernatantand the supernatant was then decanted. The solid phase in each tube waswashed once with 1 ml of water. The solid phase was then resuspended in100 ul water and counted as described in Example 12B below. Table 1shows the results obtained. In Table 1 T represents the total RLU ofeach conjugate added, B_(o) represents the total RLU associated with thesolid phase in the final resuspension, for each conjugate, in theabsence of any Vitamin B₁₂, and B_(o) /T is the percentage of the totalRLU added which were associated with the solid phase, for eachconjugate.

                  TABLE 1                                                         ______________________________________                                        Conjugate                                                                              T            B.sub.o   B.sub.o /T %                                  ______________________________________                                        1          18 × 10.sup.6                                                                      3.8 × 10.sup.4                                                                    0.21                                          2          29 × 10.sup.6                                                                        5 × 10.sup.4                                                                    0.17                                          3        17.5 × 10.sup.6                                                                      7.7 × 10.sup.4                                                                    0.44                                          4        15.2 × 10.sup.6                                                                      6.6 × 10.sup.4                                                                    0.44                                          5        20.6 × 10.sup.6                                                                      26.1 × 10.sup.4                                                                   1.27                                          6          12 × 10.sup.6                                                                       20 × 10.sup.4                                                                    1.67                                          ______________________________________                                    

FIG. 7 is a plot of B/B_(o) against Vitamin B₁₂ concentration for eachof the conjugates. B represents the total RLU associated with the solidphase in the final resuspension for a particular concentration ofVitamin B₁₂ in the sample and B_(o) is as described above. The conjugatethat performed the best (highest Bo/T value) came from the lower band(conjugate #6) which originated from monocarboxylic Vitamin B₁₂ III.

100 ug of the B₁₂ -ED-DMAE conjugate #6 was separated on an analyticalHPLC system as described in Example 5D eluted with mixture of 0.05Mtriethylammonium acetate, pH 4.5 (Solvent A) and acetonitrile (SolventB) in linear gradient from 40% B/60% A to 50% B/50% A over a 10 min.period. The flow rate of the-eluent was 1 ml/min. and the elutedmaterials were detected at 260 nm. The chromatogram revealed thepresence of two peaks (retention times of 5.66 min. and 7.86 min.).These two peaks when isolated and evaluated separately gave identicalassay performance whether combined or alone.

EXAMPLE 7 Preparation of N-Trifluoroacetyl-Folic Acid (TFA-Fol)

A mixture of folic acid (1.0 g, 2.27 mmole) and trifluoroaceticanhydride (2 ml, 6.4 mmole) was stirred at room temperature for 1 hourand then evaporated under vacuo. The residue from the evaporation wastriturated with a minimal amount of methanol and the supernatant removedby filtration. The resultant wet cake was evaporated to dryness toproduce TFA-Fol with an Rf of 0.35 when chromotographed on a TLC plate(Silica gel 60, Merck & Co.) using chloroform/methanol/water (55:40:5).

EXAMPLE 8

Preparation of Folate-SulfoCys-ED-DMAE conjugate

A solution of TFA-Fol (27 mg, 0.05 mmole) (Example 7) in 3.75 ml of DMFwas diluted with 1.8 ml of chloroform, cooled in an ice bath and treatedwith triethylamine (0.065 ml, 0.45 mmole) and ethyl chloroformate (0.03ml, 0.3 mmole) to form a reaction mixture. After 30 min. of stirring,the reaction mixture was evaporated to dryness under vacuo. To theresidue which resulted from the evaporation were added 4.5 ml ofDMF/chloroform (2:1), SulfoCys-ED-DMAE (31 mg, 0.04 mmole) andtriethylamine (0.035 ml, 0.24 mmole) to form a second reaction mixture.The second reaction mixture was stirred at room temperature overnightand evaporated to form a second residue.

The second residue was purified on a 10×20 cm silica gel preparative TLCplate (Silica gel 60, Merck & Co.) developed withchloroform/methanol/water (55:40:5).

The yellow band which developed at about an Rf of 0.64 was stripped andeluted with the same solvent system. The eluent was evaporated toproduce crude TFA-Fol-SulfoCys-ED-DMAE (11 mg). TLC analysis of theproduct showed two major UV positive spots (Rf of 0.6 and 0.5). The spothaving Rf of 0.5 was not affected by the subsequent deblockingconditions and was, therefore, considered as an undesirable contaminant.

The crude TFA-Fol-SulfoCys-ED-DMAE obtained above was treated with 200ul of 36% HBr/Acetic acid at room temperature overnight to form a thirdreaction mixture. This third reaction mixture was treated with about 10ml of anhydrous ethylether to form a precipitate. After 1 hour ofstanding, the supernatant was removed from the precipitate by carefulpipeting. The precipitate was then dissolved in about 0.5 ml ofchloroform/methanol/water (65:25:4) and purified on one 20×20 cm silicagel analytical TLC plate (Silica gel 60, Merck & Co., Inc.). The TLCplate was developed with chloroform/methanol/water (55:40:5). The yellowband which developed at Rf of 0.38 was stripped and eluted with the samedeveloping solvent system. The eluent was evaporated to produce theFolate-SulfoCys-ED-DMAE conjugate.

EXAMPLE 9 Preparation of Cortisol-3-CMO-ED-DMAE Conjugate

A solution of 3-carboxylmethyloxime-cortisol (Cortisol-3-CMO, 10 mg,0.022 mmole) (Steraloids, Wilton, N.H.) in 0.2 ml of DMF was dilutedwith 0.8 ml of chloroform, cooled in an ice bath, and treated withdicyclohexylcarbodiimide (DCC, 5.5 mg, 0.0266 mmole) in 0.2 ml ofchloroform to produce a reaction mixture. After 10 min. of stirring, thereaction mixture was treated with a solution of DMAE-ED (5.5 mg, 0.01mmole) in 0.4 ml of DMF to produce a second reaction mixture. The secondreaction mixture was stirred at room temperature overnight andevaporated under vacuo. The residue from the evaporation was purified ona 10×20 cm silica gel preparative TLC plate (Merck & Co.) and developedwith 10% methanol/chloroform. The yellow band which developed at Rf of0.28 was stripped and eluted with 20% methanol/chloroform. The eluentwas evaporated to give Cortisol-3-CMO-ED-DMAE (3.56 mg, 42%). FAB MassSpectral analysis (performed by Institute of Chemical Analysis,Northeastern Univ., Boston, Mass.) in the positive ion mode gave a M+peak of 845.

EXAMPLE 10 Preparation of Estradiol-6-CMO-ED-DMAE Conjugate

A solution of 6-carboxymethyloxime-17-beta-estradiol (Estradiol-6-CMO)(23.1 mg, 0.062 mmole) (Steraloids, Wilton, N.H.) in 2 ml of DMF/CHCl₃(1:1) was cooled in an ice bath, and treated with DCC (15.4 mg, 0.074mmole) to produce a reaction mixture. After 10 min. of stirring, thereaction mixture was treated with DMAE-ED (30.1 mg, 0.059 mmole) toproduce a second reaction mixture. The second reaction mixture wasstirred at room temperature overnight and evaporated under vacuo. Theresidue from the evaporation was purified on one 20×20 cm silica gelpreparative TLC plate developed with 5% methanol/chloroform. The yellowband which developed at Rf of 0.17 was stripped and eluted with 20%methanol/chloroform. The eluent was evaporated to giveEstradiol-6-CMO-ED-DMAE (11.9 mg, 26%). FAB Mass Spectral analysis(performed by Institute of Chemical Analysis, Northeastern University,Boston, Mass.) in the positive ion mode gave a M+ peak of 789.

EXAMPLE 11 Preparation of Thromboxane B₂ -ED-DMAE Conjugate (TxB₂-ED-DMAE)

A solution of Thromboxane B₂ (TxB2) (2.5 mg, 0.0067 mmole) (Biomol,Plymouth Meeting, Pa.) in 0.4 ml of DMF/CHCl₃ (1:1) was cooled in an icebath, treated with triethylamine (6 ul, 0.04 mmole), and ethylchloroformate (2 ul, 0.02 mmole) to produce a reaction mixture. After 30min. of stirring, the reactionmixture was evaporated to dryness. Theresidue from the evaporation was dissolved in 0.4 ml of DMF/CHCl₃ (1:1),treated with triethylamine (6 ul, 0.04 mmole) and DMAE-ED (4.5 mg, 0.009mmole) to produce a second reaction mixture. The second reaction mixturewas stirred at room temperature and evaporated under vacuo to form asecond residue. The second residue was taken up in about 0.5 ml ofchloroform/methanol/water (65:25:4) and purified on a 20×20 cm silicagel analytical TLC plate (Merck & Co.) developed with 15%methanol/chloroform.

The major yellow band which developed at Rf of 0.49 was stripped andeluted with 15% methanol/chloroform. The eluent was evaporated toproduce TxB₂ -ED-DMAE.

EXAMPLE 12 Vitamin B₁₂ Assay

A. Preparation of Intrinsic Factor Paramagnetic Particles (IF-PMP)

PMP (obtained from Advanced Magnetics Inc., Cambridge, Mass.) wereactivated with glutaraldehyde as described in U.S. Pat. No. 4,454,083.

To a solution of human serum albumin (HSA) (400 mg, Immunosearch, TomsRiver, N.J.) in 25 ml of 10 mM sodium phosphate, pH 7.4, was addedpurified hog Intrinsic Factor (purchased from Dr. R. H. Allen,University of Colorado Medical Center, Denver, Colo.) (75 ug) in 5 ml ofsaline to produce a protein mixture.

The protein mixture was added to a suspension of the activated PMP (5 g)in 60 ml of 10 mM sodium phosphate and shaken at room temperatureovernight to produce IF-PMP.

The IF-PMP were then washed and the excessive activated groups werequenched with glycine.

The IF-PMP were resuspended in 200 ml of 30 mM PBS with 0.1% sodiumazide, 0.1% BSA and 0.001% BgG, cured at 50° C. for 16 hrs, washed threetimes with 10 mM sodium phosphate, washed three times with GlycineBuffer (0.325 glycine, 0.1% sodium azide, and 0.1% BSA, pH 7.8),resuspended in the Glycine Buffer (25 mg/ml) and stored at 4° C. untilneeded.

B. Simultaneous Assay

A series of standards in 6% HSA (in 120 mM PBS with 0.2% sodium azideand 0.4 g/l merthiolate) with known increasing amounts of Vitamin B₁₂,were added to 12×75 mm plastic tubes (100 ul/tube). 0.1 ml of ReleasingAgent (0.5M NaOH, 50 ug/ml KCN, 0.3 ug/ml cobinamide, 0.064Mdithiothreitol) was added to the tubes and the tubes were incubated atroom temperature for 15 minutes. The IF-PMP prepared in A was diluted1:312 in the Glycine Buffer (80 ug/ml). 0.5 ml of the diluted IF-PMP (40ug/tube) and 0.1 ml of the B₁₂ -ED-DMAE conjugate #6 prepared in Example6, diluted in PBS with 0.1% BSA and 0.1% sodium azide (4×10⁶ RLU/tube),were then added to the tubes and the tubes were incubated for 60 min. atroom temperature.

The tubes were then placed in a magnetic rack useful for magneticseparation of paramagnetic particles in tubes (available from CibaCorning Diagnostics Corp., Medfield, Mass.). The magnetic fieldseparated the particles from the supernatant and the supernatant wasthen decanted. The particles were washed once in 1 ml of water,vortexed, magnetically separated from the wash and decanted. Theparticles were then resuspended in 0.1 ml of a 1 mM ethyl maleimide.

The tubes were then placed in a luminometer (MAGICR LITE Analyzer, CibaCorning Diagnostics Corp., Medfield, Mass.). 0.3 ml of a solution of0.5% hydrogen peroxide in 0.1 N HN0₃ was added to each tube by theluminometer and the light emission was triggered by the addition of 0.3ml of 0.25N NaOH containing ARQUAD surfactant (Armack Chemicals,Chicago, Ill.). The measured RLU's of each tube normalized against theRLU's of the zero standard were plotted against their respective VitaminB₁₂ concentrations as shown in FIG. 8.

C. Split Incubation Assay

A series of Vitamin B₁₂ standards in 5% HSA (in PBS with 0.2% sodiumazide, 2 mg/l amphotericin and 24 mg/l gentamycin) with known increasingamounts of Vitamin B₁₂ were added to tubes (100 ul/tube) and incubatedwith the Releasing Agent of B except that the cobinamide was omitted.0.5 ml of the diluted IF-PMP of B (40 ug/tube), but with 0.06 ug/mlcobinamide added to the buffer, was added to each tube and the tubeswere then incubated for 45 minutes at room temperature. 0.1 ml of theB₁₂ -ED-DMAE conjugate #6 (8×10⁶ RLU) prepared in Example 6 diluted with10 mM PBS, pH 7.4, containing 0.1% sodium azide and 0.1% BSA, was thenadded to each tube and the tubes were then incubated for 30 minutes atroom temperature. The particles in the tubes were magneticallyseparated, washed, resuspended, and counted as described in B. Themeasured RLU's normalized against the RLU's of the zero standard foreach tube were plotted against their respective Vitamin B₁₂concentration as shown in FIG. 9.

EXAMPLE 13 Folate Assay

A. Reagents

The standards used in the Folate assay were PGA (pteroylglutamic acid)(Sigma Chemical Co, St. Louis, Mo.) dissolved in 120mM PBS, pH 7.4 with4% HSA, 0.2% sodium azide, 2 mg/l amphotericin, and 24 mg/1 gentamycinadded as preservatives. The folate concentrations were zero, 0.25, 0.5,1.0, 2.5, 5, 10, 15, 20, and 30 ng PGA/ml.

The Releasing Agent was 0.5N NaOH containing 64 mM dithiothreitol.

Folate-SulfoCys-ED-DMAE conjugate (8.8×10¹¹ RLU) obtained in Example 8was first dissolved in 22 ml of 10% DMF/water. The solution was thenfurther diluted 1:11250 with 325 mM glycine containing 0.1% BSA and 0.1%sodium azide to form a second solution. This second solution wasfiltered through a 0.2 um cellulose acetate filter (Schleicher andSchuell, Keene, N.H.) to produce a tracer solution. 500 ul of the tracersolution was added per test.

The binder in the assay was Folate Binding Protein (FBP) (a bovine milklactoglobulin, purchased from Dr. R. H. Allen, University of ColoradoMedical Center, Denver, Colo.). FBP-PMP and Bovine Gamma Globulin(BgG)-PMP were prepared by the method described in U.S. Pat. No.4,454,088. The FBP-PMP (0.96 mg/ml) was diluted 1:60 in 10 mM PBS with0.1% BSA and 0.1% sodium azide, pH 7.4. This was bulked with BgG-PMP at0.4 mg/ml to form the solid phase binder. 100 ul of this solid phasebinder was added per test, resulting in the addition of 1.6 ug FBP-PMPand 40 ug BgG PMP. In the final assay there was 100 ul of sample orstandard, 100 ul of Releasing Agent, 500 ul of tracer solution and 100ul of solid phase binder, for a total assay volume of 800 ul.

B. Assay Procedure

Standards or samples (100 ul) were added to 12×75 mm polystyrene tubes(Sarstedt, West Germany). To each tube was added 100 ul of the ReleasingAgent of A. The tubes were vortexed and incubated for 15 min. at roomtemperature. The tracer solution of A (500 ul) was then added to eachtube, followed by the addition of the solid phase binder of A (100 ul).The tubes were vortexed again and incubated for one hour at roomtemperature. The tubes were then put on a magnetic separator for 3minutes, decanted, and blotted. 1 ml of deionized water was added toeach tube to wash out excess unbound tracer. The solid phase in thetubes was magnetically separated for 3 min., the supernatant decanted,and the tubes drained for 3 minutes. To the resulting pellets in thetubes was added 100 ul of 1 mM ethyl maleimide. The tubes were thenplaced in a luminometer and counted as described in Example 12B. TheRLU's for each standard were normalized against the RLU's of the zerostandard and plotted against the respective folate concentration of thestandards to give a displacement curve as shown in FIG. 10.

EXAMPLE 14 Cortisol Assay

A. Reagent Preparation

The Cortisol-ED-DMAE conjugate of Example 9 was dissolved in methanoland kept at -20° C. as a stock solution. The final cortisol-ED-DMAEconjugate was diluted in a buffer containing 10 mM sodium phosphate, pH7.4, 0.1% bovine serum albumin, 0.4 mg/ml of8-anilino-1-naphthalenesulphonate, 0.1% Triton X-100, and 0.05% sodiumazide, to produce the tracer solution.

Rabbit anti-cortisol antiserum was bought from Bioclinical Group,Cambridge, Mass. The antibody was immobilized on PMP (Advanced MagneticsInc.) as described in U.S. Pat. No. 4,554,088 except that 0.01 M sodiumacetate buffer, pH 5.5, was used instead of 0.1M sodium phosphatebuffer, pH 7.4. The final PMP wet cake was diluted with a buffercontaining 0.01M sodium phosphate, 0.1% bovine serum albumin, 4 ng/ml11-deoxycortisol, and 0.4 mg/ml 8-anilino-1-naphthalene sulfonate, pH7.4, to form a PMP suspension (10 mg/ml).

B. Assay Procedure

25 ml each of cortisol standards with concentrations from 0 to 750 ng/mlwere added to 12×75 mm polystyrene test tubes (Sarstedt, West Germany)in duplicate. 100 ul of the tracer solution of A with total activity of10⁶ RLU were then added to the tubes followed by 500 ul of the dilutedPMP suspension of A. After vortexing, the tubes were incubated for 1hour at room temperature. The PMP in the tubes were magneticallyseparated from the supernatant. The supernatant in each tube was thendecanted and the PMP in each tube were washed once with 500 ul of 0.87%saline and then resuspended in 100 ul of water. The tubes were thenplaced in a luminometer and counted as described in Example 12B. Astandard curve in FIG. 11 shows the displacement of tracer bound to PMPby added cortisol in the standard. The displacement is inverselyproportional to the concentration of the cortisol in the standard.

EXAMPLE 15 Estradiol Assay

A. Reagent Preparation

The Estradiol-ED-DMAE conjugate of Example 10 was dissolved in methanoland kept at -20° C. as a stock solution. The stock solution was dilutedwith 0.01M sodium phosphate buffer, pH 7.4, containing 0.1% bovine serumalbumin, 0.15M NaCl, and 0.05% sodium azide to form a tracer solution.

Monoclonal anti-estradiol antibody was produced in mice (A/J) byimmunization with a BSA-estradiol conjugate and subsequent fusion of thesplenocytes with Sp2/0-Ag14 myeloma cells by the procedure described byKohler and Milstein in Nature (London), vol. 256, pp. 495-497 (1975).Hybridoma cells secreting anti-estradiol antibody were detected by thefollowing procedure: Supernatant from the cells were diluted 1:5 inphosphate buffered saline containing 1 mg/ml bovine serum albumin. 10 ulof each diluted supernatant and 100 ul of acridinium ester-labelledestradiol-fowl gamma globulin as tracer were added to a test tube andincubated for one hour at room temperature. Goat anti-mouse IgG coupledto paramagnetic particles were added to each tube and incubated furtherfor 10 minutes at room temperature. The particles were magneticallyseparated and read on a luminometer for tracer bound to the particles.The cells that tested positive (i.e., produce photon counts overbackground) were plated at 0.1 cell/well and retested after growth.

Cells resulting from this regrowth which tested positive were theninjected intraperitoneally into pristane-primed mice (CAF,). Asciticfluid from these mice was collected after 3-5 weeks. The anti-estradiolantibody was used directly without further purification. Goat anti-mouseIgG PMP particles were prepared by immobilizing the IgG fraction of goatanti-mouse IgG antiserum (Jackson Laboratory, Pa.) on paramagneticparticles by the method described in U.S. Pat. No. 4,454,088. The finalPMP wet cake was diluted with phosphate buffered saline containing 1mg/ml bovine serum albumin (PBS/BSA)to produce a PMP suspension with afinal concentration of 10 mg/ml.

B. Assay Procedure

50 ul each of a series of estradiol standards with concentration rangefrom 0 to 2000 pg/ml were added to 12×75 mm polystyrene test tubes(Sarstedt, West Germany) followed by the addition of 100 ul of thetracer solution of A with total activity of 385,000 RLU. 100 ul of theascitic fluid from A was diluted 1:20000 in PBS/BSA buffer and then wasadded to each tube. The tubes were all vortexed and incubated for onehour at room temperature. The PMP suspension of A was diluted in PBS/BSAto a final concentration of 80 ug/ml. 500 ul of the diluted PMPsuspension was then added to each of the test tubes. The tubes were thenvortexed and incubated for 30 minutes at room temperature. The PMP inthe tubes were then magnetically separated. The PMP in each tube wasthen washed once with 500 ul of saline containing 0.05% Triton X-100,magnetically separated, and the supernatant decanted. The PMP was thenresuspended in 100 ul of water. The tubes were then placed in aluminometer and counted as described in Example 12B. The displacementcurve in FIG. 12 shows that the photon counts are inversely proportionalto the concentration of estradiol in the standards.

EXAMPLE 16 Thromboxane B₂ (TxB₂) assay

A. Reagent preparation

The TxB-ED-DMAE conjugate of Example 11 was dissolved in methanol andkept at -80° C. as a stock solution. The stock solution was diluted with0.01M sodium phosphate buffer, pH 7.4, containing 0.1% BSA, 0.15M NaCLand 0.05% sodium azide to produce the tracer solution.

Rabbit anti-TxB₂ antiserum was bought from Cayman Chemicals Co., AnnArbor, Mich. The antiserum was diluted with 0.01M sodium phosphatebuffer, pH 7.4, containing 0.15M sodium chloride, 1 mg/ml bovine serumalbumin, 0.05% sodium azide.

Goat anti-rabbit IgG PMP was prepared by immobilizing the IgG fractionof goat anti-rabbit IgG antiserum (Jackson Laboratory, Pa.) onparamagnetic particles (PMP) by the method described in U.S. Pat. No.4,454,088. The final PMP wet cake was diluted with PBS/BSA buffer (10mg/ml) to produce a PMP suspension.

B. Assay procedure

100 ul of each of a series of TxB₂ standards in PBS/BSA (0-30 ng/ml)were added to polystyrene test tubes (12×75 mm, Sarstedt, West Germany).10 ul of the tracer solution of A with a total activity of 350,000 RLU,was then added to each tube. 10 ul of rabbit anti-TxB₂ antiserum(prepared in A diluted 1/40000 in PBS/BSA) was pipeted into all tubes.All tubes were vortexed and incubated at room temperature for 1 hour.500 ul of the diluted PMP suspension of A was then added to all thetubes and incubated for 45 minutes at room temperature. The PMP in thetubes were then magnetically separated from the supernatant. Thesupernatant was decanted and the PMP were then washed once with 500 ulof water then resuspended in 100 ul of water. The tubes were then placedin a luminometer and counted as described in Example 12B. Thedisplacement curve in FIG. 13 shows that the photon counts wereinversely proportional to the concentration of TxB₂ in the standards.

What is claimed is:
 1. A luminescent conjugate comprising an acridiniumester bound to a specific binding member where the acridinium ester hasthe formula: ##STR21## wherein: X⁻ is an anion;R₁ is (a) attached to thering nitrogen of the acridine nucleus of the acridinium ester through acarbon, (b) comprises up to 20 heteroatoms, and (c) is selected from thegroup consisting of alkenyl, alkyl, alkynyl, aralkyl, and aryl; R₂, R₃,R₅, and R₇ are selected from the group consisting of alkoxyl, amino,halide, hydrogen, hydroxyl, nitro, --CN, ##STR22## --SCN, --COOH, and--SO₃ H; R₄ and R₈ are selected from the group consisting of alkenyl,alkoxyl, alkyl, alkynyl, and aralkyl; R₆ is selected from the groupconsisting of Q--R--Nu, ##STR23## and Q--Nu where: Nu is a nucleophilicgroup; Q is selected from the group consisting of diazo, ##STR24## and--NH--; and I is selected from the group consisting of --SO₃ H, --OSO₃H, --PO(OH)₂, --COOH, and --OPO(OH)₂ ;except that, where R₆ ═QNu, thecondition where Q═ ##STR25## and Nu═--OH is excluded; and R comprises upto 20 heteroatoms and is selected from the group consisting of alkenyl,alkyl, alkynyl, aralkyl, and aryl.
 2. The luminescent conjugate of claim1 wherein:X⁻ is selected from the group consisting of halide, CH₃ SO₄ ⁻,FSO₃ ⁻, CF₃ SO₃ ⁻, C₄ F₉ SO₃ ⁻, and ##STR26## R₁ (a) comprises 1 to 24carbon atoms, (b) comprises up to 20 heteroatoms selected from the groupconsisting of nitrogen, oxygen, phosphorous, and sulfur, and (c) isselected from the group consisting of alkenyl, alkyl, alkynyl, and aryl;R₂, R₃, R₅, and R₇ are selected from the group consisting of C₁ -C₄alkoxyl, amino, halide, hydrogen, hydroxyl, nitro, --CN, --SCN, --COOH,and --SO₃ H; R₄ and R₈ are (a) selected from the group consisting ofalkenyl, alkoxyl, alkyl, and alkynyl, and (b) comprise 1 to 8 carbonatoms; Nu is selected from the group consisting of an amino group, ahydroxyl group, a sulfhydryl group, an organo-metallic moiety, and anactive methylene group; and R (a) comprises 1 to 24 carbon atoms, and(b) comprises up to 20 heteroatoms selected from the group consisting ofnitrogen, oxygen, phosphorous, and sulfur.
 3. The luminescent conjugateof claim 1 wherein:X⁻ is a halide; R₁ is an alkyl comprising 1 to 10carbon atoms; R₂, R₃, R₅, and R₇ are selected from the group consistingof C₁ -C₄ alkoxyl, amino, hydrogen, nitro, --CN, and --SO₃ H; R₄ and R₈are an alkyl comprising 1 to 4 carbon atoms; and Nu is selected from thegroup consisting of an amino group, a hydroxyl group, a sulfhydrylgroup, an organo-metallic moiety selected from the group consisting ofGrignard reagents, lithium compounds, and phenylsodium, and an activemethylene group adjacent to a strong electron-withdrawing group selectedfrom the group consisting of --NO₂, --CN, --SO₃ H, --N(R)₃ ⁺, and--S(R)₃ ³⁰ .
 4. The luminescent conjugate of claim 1 wherein:X⁻ isbromide; R₁ is methyl; R₂, R₃, R₅, and R₇ are hydrogen; and R₄ and R₈are methyl.
 5. The luminescent conjugate of claim 1 wherein R.sub.₆ is--CONH--CH₂ CH₂ --NH₂.
 6. The luminescent conjugate of claim 1 whereinR₆ is ##STR27##
 7. The luminescent conjugate of claim 1, 2, 3, 4, 5, or6 wherein the specific binding member is selected from the groupconsisting of an analyte, an analyte analog and a complementary binderwhich specifically binds to or hybridizes to both the analyte and theanalyte analog.
 8. The luminescent conjugate of claim 7 wherein thespecific binding member is an antigen, a hapten, or a ligand.
 9. Theluminescent conjugate of claim 7 wherein the specific binding member isa protein, a nucleic acid, or a molecule comprising nucleic acids. 10.The luminescent conjugate of claim 1, 2, 3, 4, or 6 wherein the specificbinding member is folate or a folate derivative.
 11. The luminescentconjugate of claim 1, 2, 3, 4, or 5 wherein the specific binding memberis Vitamin B₁₂ or a Vitamin B₁₂ derivative.
 12. The luminescentconjugate of claim 1, 2, 3, 4, or 5 wherein the specific binding memberis 17-beta-estradiol or a 17-beta-estradiol derivative.
 13. Theluminescent conjugate of claim 1, 2, 3, 4, or 5 wherein the specificbinding member is cortisol or a cortisol derivative.
 14. The luminescentconjugate of claim 1, 2, 3, 4, or 5 wherein the specific binding membera prostaglandin or a prostaglandin derivative.
 15. The luminescentconjugate of claim 14 wherein the specific binding member is thromboxaneB₂.